Antibiotic resistant bacterial infections pose a serious threat to human health and strategies to overcome these infections are desperately needed. Many clinically used antibiotics target the final steps of peptidoglycan (PG) biosynthesis, which involve the polymerization of disaccharide-peptide subunits by peptidoglycan glycosyltransferases (PGTs) and the crosslinking of the polymerized chains by transpeptidases (TPs). There are major gaps in our understanding of these steps, which has hampered efforts to develop new antibiotics. The PG matrix is assembled into a complex three-dimensional polymer from a single disaccharide substrate. In order to understand how the PGTs and TPs function, one must be able to make complicated substrates designed to discriminate between different subsites of enzymes that couple identical molecules. We propose three specific aims involving the use of peptidoglycan fragments to address major gaps in knowledge about PGTs and TPs. For example, although the TPs are the lethal targets of the beta-lactams, they remain almost completely uncharacterized. In Aim I we propose to a) identify tetrasaccharide substrates containing a blocked non-reducing end that activate PGTs for elongation, and b) to use these molecules to obtain a crystal structure of the PGT elongation complex. PGT domains we and others have previously crystallized with moenomycin will be used for these studies. A structure of an elongation competent PGT:substrate complex would provide new insights into catalysis and a new basis for virtual screening and design of inhibitors. In Aim II, we propose to a) make peptidoglycan polymer substrates capable of activation but not crosslinking, and b) to use them in conjunction with polymer substrates capable of activation and crosslinking to develop assays that report on peptide activation, hydrolysis, and crosslinking by bacterial transpeptidases. E. coli PBP1A and E. faecalis PBP2A will be used as model enzymes for these experiments. The ability to assay TP activity will make it possible to address the functions of TP-regulatory proteins in bacteria and to characterize the substrate specificities of TPs from other organisms. In Aim III, we propose to a) make the three main stem-peptide variants of S. aureus Lipid II and use these substrates to make the corresponding PG polymers; and b) to characterize the abilities of the beta-lactam sensitive and beta-lactam resistant transpeptides in MRSA to activate, hydrolyze, and crosslink these polymers. It has been proposed that the S. aureus TPs have different substrate preferences, and that this explains why deleting genes involved in stem peptide branching restores beta-lactam sensitivity to MRSA strains containing an intrinsically resistant transpeptidase. There is no biochemical evidence for this hypothesis since the substrate preferences of the S. aureus TPs have not been examined. The results of the experiments in Aim III have implications for new approaches to overcome MRSA that involve combining a beta-lactam with compounds that target other proteins involved in methicillin resistance.